1.6.14
INDUCTION OF EL13-GFP FUSIONS IN TRANSGENIC ARABIDOPSIS THALIANA BY AVRB DELIVERED BY THE HRP (TYPE III) SECRETION SYSTEM OF PSEUDOMONAS SYRINGAE

WL DENG1*, JR ALFANO2* and A COLLMER1

1Department of Plant Pathology, Cornell University, lthaca, NY 14853, USA; 2Department of Biological Sciences, University of Nevada, Las Vegas, NV 89154, USA
*Both authors contributed equally to this work.

Background and objectives
The hypersensitive response (HR) of higher plants is characterized by the rapid, localized death of plant cells at the site of invasion of an incompatible pathogen. The ability to elicit the HR or pathogenesis in host plants is conferred by hrp genes. A functional cluster of Pseudomonas syringae pv. syringae hrp genes was cloned to produce cosmid pHIR11. The saprophyte P. fluorescens carrying pHIR11 is capable of delivering avr signals from several well characterized avr genes. These data, together with other strong indirect evidence, indicate that the Hrp secretion system translocates many Avr proteins inside the plant cell. To further dissect the delivery of Avr proteins by the Hrp secretion system, we sought to develop cell biological tools that would monitor the deployment of Avr proteins and subsequent gene expression events in host cells. The ELI3 gene in Arabidopsis thaliana is specifically induced in plants carrying the resistance gene RPM1 [1] that have been infiltrated with hrp+ bacteria carrying the avirulence gene avrB.

Results and conclusions
We made transcriptional fusions of the EL13 promoter with a gene that encodes the green fluorescent protein (GFP). The gfp gene used in these studies has been previously modified to lack a cryptic intron splice site that is recognized in Arabidopsis [2]. The ELI3-gfp fusion was subcloned into a plant transformation vector, and transgenic plants were isolated using Agrobacterium-mediated transformation. ELI3-gfp-containing Arabidopsis plants emitted green fluorescence when P. fluorescens (pHIR11) carrying avrB was infiltrated into leaves, but not in the absence of avrB, indicating that the production of GFP was dependent on the presence of avrB. When pHIR11 derivatives that contained mutations in genes required for type III secretion were used in these experiments, no GFP was produced, confirming that the delivery of AvrB into plant cells is dependent on a functional Hrp secretion system. Using confocal fluorescence microscopy, we are currently analysing transgenic ELI3-gfp Arabidopsis plants to determine spatial and temporal patterns of ELI3 gene expression in relation to individual bacterial cells.

References
1. Kiedrowski S, Kawalleck P, Hahibrock K et al., 1992. EMBO Journal 11, 4677-4684.
2. Chiu WL, Niwa Y, Zeng W et al., 1996. Current Biology 6, 325-330.